The invention relates to new isolated immunoglobulins which are devoid of light polypeptide chains. These immunoglobulins do not consist in the degradation products of immunoglobulins composed of both heavy polypeptide and light polypeptide chains but to the contrary, the invention defines a new member of the family of the immunoglobulins, especially a new type of molecules capable of being involved in the immune recognition. Such immunoglobulins can be used for several purposes, especially for diagnosis or therapeutical purposes including protection against pathological agents or regulation of the expression or activity of proteins.
Up to now the structure proposed for immunoglobulins consists of a four-chain model referring to the presence of two identical light polypeptide chains (light chains) and two identical heavy polypeptide chains (heavy chains) linked together by disulfide bonds to form a y- or T-shaped macromolecules. These chains are composed of a constant region and a variable region, the constant region being subdivided in several domains. The two heavy polypeptide chains are usually linked by disulphide bounds in a so-called xe2x80x9chinge regionxe2x80x9d situated between the first and second domains of the constant region.
Among the proteins forming the class of the immunoglobulins, most of them are antibodies and accordingly present an antigen binding site or several antigen binding sites.
According to the four-chain model, the antigen binding site of an antibody is located in the variable domains of each of the heavy and light chains, and requires the association of the heavy and the light chains variable domains.
For the definition of these four-chain model immunoglobulins, reference is made to Roitt. I et al (Immunology-second-Edition Gower Medical Publishing USA, 1989). Reference is especially made to the part concerning the definition of the four-chain immunoglobulins, their polypeptidic and genetic structures, the definition of their variable and constant regions and the obtention of the fragments produced by enzymatic degradation according to well known techniques.
The inventors have surprisingly established that different molecules can be isolated from animals which naturally produce them, which molecules have functional properties of immunoglobulins these functions being in some cases related to structural elements which are distinct from those involved in the function of four-chain immunoglobulins due for instance to the absence of light chains.
The invention relates to two-chain model immunoglobulins which neither correspond to fragments obtained for instance by the degradation in particular the enzymatic degradation of a natural four-chain model immunoglobulin, nor correspond to the expression in host cells, of DNA coding for the constant or the variable region of a natural four-chain model immunoglobulin or a part of these regions, nor correspond to antibodies produced in lymphopaties for example in mice, rats or human.
E. S. Ward et al (1) have described some experiments performed on variable domains of heavy polypeptide chains (VH) or/and light polypeptide chains (VK/FV) to test the ability of these variable domains, to bind specific antigens. For this purpose, a library of VH genes was prepared from the spleen genomic DNA of mice previously immunized with these specific antigens.
Ward et al have described in their publication that VH domains are relatively sticky, presumably due to the exposed hydrophobic surface normally capped by the VK or Vxcex domains. They consequently envisage that it should be possible to design VH domains having improved properties and further that VH domains with binding activities could serve as the building blocks for making variable fragments (Fv fragments) or complete antibodies.
The invention does not start from the idea that the different fragments (light and heavy chains) and the different domains of these fragments of four-chain model immunoglobulin can be modified to define new or improved antigen binding sites or a four-chain model immunoglobulin.
The inventors have determined that immunoglobulins can have a different structure than the known four-chain model and that such different immunoglobulins offer new means for the preparation of diagnosis reagents, therapeutical agents or any other reagent for use in research or industrial purposes.
Thus the invention provides new immunoglobulins which are capable of showing functional properties of four-chain model immunoglobulins although their structure appears to be more appropriate in many circumstances for their use, their preparation and in some cases for their modification. Moreover these molecules can be considered as lead structures for the modification of other immunoglobulins. The advantages which are provided by these immunoglobulins comprise the possibility to prepare them with an increased facility.
The invention accordingly relates to immunoglobulins characterized in that they comprise two heavy polypeptide chains sufficient for the formation of a complete antigen binding site or several antigen binding sites, these immunoglobulins being further devoid of light polypeptide chains. In a particular embodiment of the invention, these immunoglobulins are further characterized by the fact that they are the product of the expression in a prokaryotic or in a eukaryotic host cell, of a DNA or of a cDNA having the sequence of an immunoglobulin devoid of light chains as obtainable from lymphocytes or other cells of Camelids.
The immunoglobulins of the invention can be obtained for example from the sequences which are described in FIG. 7.
The immunoglobulins of the invention, which are devoid of light chains are such that the variable domains of their heavy chains have properties differing from those of the four-chain immunoglobulin VH. The variable domain of a heavy-chain immunoglobulin of the invention has no normal interaction sites with the VL or with the CH1 domain which do not exist in the heavy chain immunoglobulins. it is hence a novel fragment in many of its properties such as solubility and position of the binding site. For clarity reasons we will call it VHH in this text to distinguish it from the classical VH of four-chain immunoglobulins.
By xe2x80x9ca complete antigen binding sitexe2x80x9d it is meant according to the invention, a site which will alone allow the recognition and complete binding of an antigen. This could be verified by any known method regarding the testing of the binding affinity.
These immunoglobulins which can be prepared by the technique of recombinant DNA, or isolated from animals, will be sometimes called xe2x80x9cheavy-chain immunoglobulinsxe2x80x9d in the following pages. In a preferred embodiment of the invention, these immunoglobulins are in a pure form.
In a first embodiment, the immunoglobulins of the invention are obtainable in prokaryotic cells, especially in E. coli cells by a process comprising the steps of:
a) cloning in a Bluecript vector of a DNA or cDNA sequence coding for the VHH domain of an immunoglobulin devoid of light chain obtainable for instance from lymphocytes of Camelids,
b) recovering the cloned fragment after amplification using a 5xe2x80x2 primer containing an Xho site and a 3xe2x80x2 primer containing the Spe site having the following sequence TC TTA ACT AGT GAG GAG ACG GTG ACC TG SEQ ID NO: 51,
c) cloning the-recovered fragment in phase in the immuno PBS vector after digestion of the vector with Xho and Spe restriction enzymes,
d) transforming host cells, especially E. coli by transfection with the recombinant immuno PBS vector of step c,
e) recovering the expression product of the VHH coding sequence, for instance by using antibodies raised against the dromadary VHH domain.
In another embodiment the immunoglobulins are hetero-specific immunoglobulins obtainable by a process comprising the steps of:
obtaining a first DNA or cDNA sequence coding for a VHH domain or part thereof having a determined specificity against a given antigen and comprised between Xho and Spe sites,
obtaining a second DNA or cDNA sequence coding for a VHH domain or part thereof, having a determined specificity different from the specificity of the first DNA or cDNA sequence and comprised between the Spe and EcoRI sites,
digesting an immuno PBS vector with EcoRI and XhoI restriction enzymes,
ligating the obtained DNA or cDNA sequences coding for VHH domains, so that the DNA or cDNA sequences are serially cloned in the vector,
transforming a host cell, especially E. coli cell by transfection, and recovering the obtained immunoglobulins.
In another embodiment, the immunoglobulins are obtainable by a process comprising the steps of:
obtaining a DNA or cDNA sequence coding for a VHH domain or part thereof, having a determined specific antigen binding site,
amplifying the obtained DNA or cDNA, using a 5xe2x80x2 primer containing an initiation codon and a HindIII site, and a 3xe2x80x2 primer containing a termination codon having a XhoI site,
recombining the amplified DNA or cDNA into the HindIII (position 2650) and XhoI (position 4067) sites of a plasmid pMM984,
transfecting permissive cells especially NB-E cells with the recombinant plasmid,
recovering the obtained products.
Successful expression can be verified with antibodies directed against a region of a VHH domain, especially by an ELISA assay.
According to another particular embodiment of this process, the immunoglobulins are cloned in a parvovirus.
In another example these immunoglobulins are obtainable by a process comprising the further cloning of a second DNA or cDNA sequence having another determined antigen binding site, in the pMM984 plasmid.
Such an Immunoglobulin can be further characterized in that it is obtainable by a process wherein the vector is Yep 52 and the transformed recombinant cell is a yeast especially S. cerevisiae. 
A particular immunoglobulin is characterized in that it has a catalytic activity, especially in that it is directed against an antigen mimicking an activated state of a given substrate. These catalytic antibodies can be modified at the level of their biding site, by random or directed mutagenesis in order to increase oe modify their catalytic function. Reference may be made to the publication of Lerner et al (TIBS November 1987. 427-430) for the general technique for the preparation of such catalytic immunoglobulins.
According to a preferred embodiment, the immunoglobulins of the invention are characterized in that their variable regions contain in position 45, an amino-acid which is different from leucine, proline or glutamine residue.
Moreover the heavy-chain immunoglobulins are not products characteristic of lymphocytes of animals nor from lymphocytes of a human patient suffering from lymphopathies. Such immunoglobulins produced in lymphopathies are monoclonal in origin and result from pathogenic mutations at the genomic level. They have apparently no antigen binding site.
The two heavy polypeptide chains of these immunoglobulins can be linked by a hinge region according to the definition of Roitt et al.
In a particular embodiment of the invention, immunoglobulins corresponding to the above-defined molecules are capable of acting as antibodies.
The antigen binding site(s) of the immunoglobulins of the invention are located in the variable region of the heavy chain.
In a particular group of these immunoglobulins each heavy polypeptide chain contains one antigen binding site on its variable region, and these sites correspond to the same amino-acid sequence.
In a further embodiment of the invention the immunoglobulins are characterized in that their heavy polypeptide chains contain a variable region (VHH) and a constant region (CH) according to the definition of Roitt et al, but are devoid of the first domain of their constant region. This first domain of the constant region is called CH1.
These immunoglobulins having no CH1 domain are such that the variable region of their chains is directly linked to the hinge region at the C-terminal part of the variable region.
The immunoglobulins of the type described here-above can comprise type G immunoglobulins and especially immunoglobulins which are defined as immunoglobulins of class 2 (IgG2) or immunoglobulins of class 3 (IgG3).
The absence of the light chain and of the first constant domain lead to a modification of the nomenclature of the immunoglobulin fragments obtained by enzymatic digestion, according to Roitt et al.
The terms Fc and pFc on the one hand, Fcxe2x80x2 and pFcxe2x80x2 on the other hand corresponding respectively to the papain and pepsin digestion fragments are maintained.
The terms Fab F(ab)2 F(abxe2x80x2)2 Fabc, Fd and Fv are no longer applicable in their original sense as these fragments have either a light chain, the variable part of the light chain or the CH1 domain.
The fragments obtained by papain digestion and composed of the VHH domain and the hinge region will be called FVHHh or F(VHHh)2 depending upon whether or not they remain linked by the disulphide bonds.
In another embodiment of the invention, immunoglobulins replying to the hereabove given definitions can be originating from animals especially from animals of the camelid family. The inventors have found out that the heavy-chain immunoglobulins which are present in camelids are not associated with a pathological situation which would induce the production of abnormal antibodies with respect to the four-chain immunoglobulins. On the basis of a comparative study of old world camelids (Camelus bactrianus and Camelus dromaderius) and new world camelids (for example Lama Paccos, Lama Glama, and Lama Vicuqna) the inventors have shown that the immunoglobulins of the invention, which are devoid of light polypeptide chains are found in all species. Nevertheless differences may be apparent in molecular weight of these immunoglobulins depending on the animals. Especially the molecular weight of a heavy chain contained in these immunoglobulins can be from approximately 43 kd to approximately 47 kd, in particular 45 kd.
Advantageously the heavy-chain immunoglobulins of the invention are secreted in blood of camelids.
Immunoglobulins according to this particular embodiment of the invention are obtainable by purification from serum of camelids and a process for the purification is described in details in the examples. In the case where the immunoglobulins are obtained from Camelids, the invention relates to immunoglobulins which are not in their natural biological environment.
According to the invention immunoglobulin IgG2 as obtainable by purification from the serum of camelids can be characterized in that:
it is not adsorbed by chromatography on Protein G Sepharose column,
it is adsorbed by chromatography on Protein A Sepharose column,
it has a molecular weight of around 100 kd after elution with a pH 4.5 buffer (0.15 M NaCl, 0.58% acetic acid adjusted to pH 4.5 by NaOH),
it consists of heavy xcex32 polypeptide chains of a molecular weight of around 46 kd preferably 45 after reduction.
According to a further embodiment of the invention another group of immunoglobulins corresponding to IgG3, as obtainable by purification from the serum of Camelids is characterized in that the immunoglobulin:
is adsorbed by chromatography on a Protein A Sepharose column,
has a molecular weight of around 100 kd after elution with a pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid),
is adsorbed by chromatography on a Protein G Sepharose column and eluted with pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid).
consists of heavy xcex33 polypeptide chains of a molecular weight of around 45 Kd in particular between 43 and 47 kd after reduction.
The immunoglobulins of the invention which are devoid of light chains, nevertheless comprise on their heavy chains a constant region and a variable region. The constant region comprises different domains.
The variable region of immunoglobulins of the invention comprises frameworks (FW) and complementarity determining regions (CDR), especially 4 frameworks and 3 complementarity regions. It is distinguished from the four-chain immunoglobulins especially by the fact that this variable region can itself contain an antigen binding site or several, without contribution of the variable region of a light chain which is absent.
The amino-acid sequences of frameworks 1 and 4 comprise among others respectively amino-acid sequences which can be selected from the following:
for the framework 1 domain
GGSVQTGGSLRLSCEISGLTFD SEQ ID NO: 1
GGSVQTGGSLRLSCAVSGFSFS SEQ ID NO: 2
GGSEQGGGSLRLSCAISGYTYG SEQ ID NO: 3
GGSVQPGGSLTLSCTVSGATYS SEQ ID NO: 4
CGSVQAGGSLRLSCTGSGFPYS SEQ ID NO: 5
GGSVQAGGSLRLSCVAGFGTS SEQ ID NO: 6
GGSVQAGGSLRLSCVSFSPSS SEQ ID NO: 7
for the framework 4 domain
WGQGTQVTVSS SEQ ID NO: 8
WGQGTLVTVSS SEQ ID NO: 9
WGQGAQVTVSS SEQ ID NO: 10
WGQGTQVTASS SEQ ID NO: 11
RGQGTQVTVSL SEQ ID NO: 12
for the CDR3 domain
As stated above, the immunoglobulins of the invention are preferably devoid of the totality of their CH1 domain.
Such immunoglobulins comprise CH2 and CH3 domains in the C-terminal region with respect to the hinge region.
According to a particular embodiment of the invention the constant region of the immunoglobulins comprises CH2 and CH3 domains comprising an amino-acid sequence selected from the following:
for the CH2 domain:
APELLGGPTVFIFPPKPKDVLSITLTP SEQ ID NO: 31
APELPGGPSVFVFPTKPKDVLSISGRP SEQ ID NO: 32
APELPGGPSVFVFPPKPKDVLSISGRP SEQ ID NO: 33
APELLGGPSVFIFPPKPKDVLSISGRP SEQ ID NO: 34
for the CH3 domain:
GQTREPQVYTLA SEQ ID NO: 35
GQTREPQVYTLAPXRLEL SEQ ID NO: 36
GQPREPQVYTLPPSRDEL SEQ ID NO: 109
GQPREPQVYTLPPSREEM SEQ ID NO: 110
GQPREPQVYTLPPSQEEM SEQ ID NO: 111
Interestingly the inventors have shown that the hinge region of the immunoglobulins of the invention can present variable lengths. When these immunoglobulins act as antibodies, the length of the hinge region will participate to the determination of the distance separating the antigen binding sites.
Preferably an immunoglobulin according to the invention is characterized in that its hinge region comprises from 0 to 50 amino-acids.
Particular sequences of hinge region of the immunoglobulins of the invention are the following.
GTNEVCKCPKCP SEQ ID NO: 37
or,
EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP SEQ ID NO: 38
The short hinge region corresponds to an IgG3 molecule and the long hinge sequence corresponds to an IgG2 molecule.
Isolated VHH derived from heavy chain immunoglobulins or VHH libraries corresponding to the heavy chain immunoglobulins can be distinguished from VHH cloning of four-chain model immunoglobulins on the basis of sequence features characterizing heavy chain immunoglobulins.
The camel heavyxe2x80x94chain immunoglobulin VHH region shows a number of differences with the VHH regions derived from 4-chain immunoglobulins from all species examined. At the levels of the residues involved in the VHH/VL interactions, an important difference is noted at the level of position 45 (FW) which is practically always leucine in the 4-chain immunoglobulins (98%), the other amino acids at this position being proline (1%) or glutamine (1%).
In the camel heavy-chain immunoglobulin, in the sequences examined at present, leucine at position 45 is only found once. It could originate from a four-chain immunoglobulin. In the other cases, it is replaced by arginine, cysteine or glutamic acid residue. The presence of charged amino acids at this position should contribute to making the VHH more soluble.
The replacement by camelid specific residues such as those of position 45 appears to be interesting for the construction of engineered VHH regions derived from the VHH repertoire of 4-chain immunoglobulins.
A second feature specific of the camelid VHH domain is the frequent presence of a cysteine in the CDR3 region associated with a cysteine in the CDR1 position 31 or 33 or FW2 region at position 45. The possibility of establishing a disulphide bond between the CDR3 region and the rest of the variable domain would contribute to the stability and positioning of the binding site.
With the exception of a single pathogenic myeloma protein (DAW) such a disulphide bond has never been encountered in immunoglobulin V regions derived from 4 chain immunoglobulins.
The heavy-chain immunoglobulins of the invention have further the particular advantage of being not sticky. Accordingly these immunoglobulins being present in the serum, aggregate much less than isolated heavy chains of a four-chain immunoglobulins. The immunoglobulins of the invention are soluble to a concentration above 0.5 mg/ml, preferably above 1 mg/ml and more advantageously above 2 mg/ml.
These immunoglobulins further bear an extensive antigen binding repertoire and undergo affinity and specificity maturation in vivo. Accordingly they allow the isolation and the preparation of antibodies having defined specificity, regarding determined antigens.
Another interesting property of the immunoglobulins of the invention is that they can be modified and especially humanized. Especially it is possible to replace all or part of the constant region of these immunoglobulins by all or part of a constant region of a human antibody. For example the CH2 and/or CH3 domains of the immunoglobulin could be replaced by the CH2 and/or CH3 domains of the IgG xcex33 human immunoglobulin.
In such humanized antibodies it is also possible to replace a part of the variable sequence, namely one or more of the framework residues which do not intervene in the binding site by human framework residues, or by a part of a human antibody.
Conversely features (especially peptide fragments) of heavy-chain immunoglobulin VHH regions, could be introduced into the VH or VL regions derived from four-chain immunoglobulins with for instance the aim of achieving greater solubility of the immunoglobulins.
The invention further relates to a fragment of an immunoglobulin which has been described hereabove and especially to a fragment selected from the following group:
a fragment corresponding to one heavy polypeptide chain of an immunoglobulin devoid of light chains,
fragments obtained by enzymatic digestion of the immunoglobulins of the invention, especially those obtained by partial digestion with papain leading to the Fc fragment (constant fragment) and leading to FVHHh fragment (containing the antigen binding sites of the heavy chains) or its dimer F(VHHh)2, or a fragment obtained by further digestion with papain of the Fc fragment, leading to the pFc fragment corresponding to the C-terminal part of the Fc fragment,
homologous fragments obtained with other proteolytic enzymes,
a fragment of at least 10 preferably 20 amino acids of the variable region of the immunoglobulin, or the complete variable region, especially a fragment corresponding to the isolated VHH domains or to the VHH dimers linked to the hinge disulphide,
a fragment corresponding to the hinge region of the immunoglobulin,or to at least 6 amino acids of this hinge region,
a fragment of the hinge region comprising a repeated sequence of Pro-X,
a fragment corresponding to at least 10 preferably 20 amino acids of the constant region or to the complete constant region of the immunoglobulin.
The invention also relates to a fragment comprising a repeated sequence, Pro-X which repeated sequence contains at least 3 repeats of Pro-X, X being any amino-acid and preferably Gln (glutamine), Lys (lysine) or Glu (acide glutamique); a particular repeated fragment is composed of a 12-fold repeat of the sequence Pro-X.
Such a fragment can be advantageously used as a link between different types of molecules.
The amino-acids of the Pro-X sequence are chosen among any natural or non natural amino-acids.
The fragments can be obtained by enzymatic degradation of the immunoglobulins. They can also be obtained by expression in cells or organisms, of nucleotide sequence coding for the immunoglobulins, or they can be chemically synthesized.
The invention also relates to anti-idiotypes antibodies belonging to the heavy chain immunoglobulin classes. Such anti-idiotypes can be produced against human or animal idiotypes. A property of these anti-idiotypes is that they can be used as idiotypic vaccines, in particular for vaccination against glycoproteins or glycolipids and where the carbohydrate determines the epitope.
The invention also relates to anti-idiotypes capable of recognizing idiotypes of heavy-chain immunoglobulins.
Such anti-idiotype antibodies can be either syngeneic antibodies or allogenic or xenogeneic antibodies.
The invention also concerns nucleotide sequences coding for all or part of a protein which amino-acid sequence comprises a peptide sequence selected from the following:
GGSVQTHGSLRLSCEISGLTFD SEQ ID NO: 1
GGSVQTGGSLRLSCAVSGFSFS SEQ ID NO: 2
GCSEQGGGSLRLSCAISGYTYG SEQ ID NO: 3
GGSVQPGGSLTLSCTVSGATYS SEQ ID NO: 4
GGSVQAGGSLRLSCTGSGFPYS SEQ ID NO: 5
GGSVQAGGSLRLSCVAGFGTS SEQ ID NO: 6
CGSVQAGGSLRLSCVSFSPSS SEQ ID NO: 7
WGQGTQVTVSS SEQ ID NO: 8
WGQGTLVTVSS SEQ ID NO: 9
WGQGAQVTVSS SEQ ID NO: 10
WGQGTQVTASS SEQ ID NO: 11
RGQGTQVTVSL SEQ ID NO: 12
APELLGGPSVFVFPPKPKDVLSISGXPK SEQ ID NO: 39
APELPGGPSVFVFPTKPKDVLSISGRPK SEQ ID NO: 40
APELPGGPSVFVFPPKPKDVLSISGRPK SEQ ID NO: 41
APELLGGPSVFIFPPKPKDVLSISGRPK SEQ ID NO: 42
GQTREPQVYTLAPXRLEL SEQ ID NO: 36
GQPREPQVYTLPPSRDEL SEQ ID NO: 109
GQPREPQVYTLPPSREEM SEQ ID NO: 110
GQPREPQVYTLPPSQEEM SEQ ID NO: 111
VTVSSGTNEVCKCPKCPAPELPGGPSVFVFP SEQ ID NO: 43
or,
VTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCPAPELLGGPSVFIFP SEQ ID NO: 44
GTNEVCKCPKCP SEQ ID NO: 37
APELPGGPSVFVFP SEQ ID NO: 45
EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP SEQ ID NO: 38
APELLGGPSVFIFP SEQ ID NO: 46
Such nucleotide sequences can be deduced from the amino-acid sequences taking into account the deneneracy of the genetic code. They can be synthesized or isolated from cells producing immunoglobulins of the invention.
A procedure for the obtention of such DNA sequences is described in the examples.
The invention also contemplates RNA, especially mRNA sequences corresponding to these DNA sequences, and also corresponding cDNA sequences.
The nucleotide sequences of the invention can further be used for the preparation of primers appropriate for the detection in cells or screening of DNA or cDNA libraries to isolate nucleotide sequences coding for immunoglobulins of the invention.
Such nucleotide sequences can be used for the preparation of recombinant vectors and the expression of these sequences contained in the vectors by host cells especially prokaryotic cells like bacteria or also eukaryotic cells and for example CHO cells, insect cells, simian cells like Vero cells, or any other mammalian cells. Especially the fact that the immunoglobulins of he invention are devoid of light chains permits to secrete them in eukaryotic cells since there is no need to have recourse to the step consisting in the formation of the BIP protein which is required in the four-chain immunoglobulins.
The inadequacies of the known methods for producing monoclonal antibodies or immunoglobulins by recombinant DNA technology comes from the necessity in the vast majority of cases to clone simultaneously the VH and VL domains corresponding to the specific binding site of 4 chain immunoglobulins. The animals and especially camelids which produce heavy-chain immunoglobulins according to the invention, and possibly other vertebrate species are capable of producing heavy-chain immunoglobulins of which the binding site is located exclusively in the VHH domain. Unlike the few heavy-chain immunoglobulins produced in other species by chain separation or by direct cloning, the camelid heavy-chain immunoglobulins have undergone extensive maturation in vivo. Moreover their V region has naturally evolved to function in absence of the VL. They are therefore ideal for producing monoclonal antibodies by recombinant DNA technology. As the obtention of specific antigen binding clones does not depend on a stochastic process necessitating a very large number of recombinant cells, this allows also a much more extensive examination of the repertoire.
This can be done at the level of the non rearranged VHH repertoire using DNA derived from an arbitrarily chosen tissue or cell type or at the level of the rearranged VHH repertoire, using DNA obtained from B lymphocytes. More interesting however is to transcribe the mRNA from antibody producing cells and to clone the cDNA with or without prior amplification into an adequate vector. This will result in the obtention of antibodies which have already undergone affinity maturation.
The examination of a large repertoire should prove to be particularly useful in the search for antibodies with catalytic activities.
The invention thus provides libraries which can be generated in a way which includes part of the hinge sequence, the identification is simple as the hinge is directly attached to the VHH domain.
These libraries can be obtained by cloning cDNA from lymphoid cells with or without prior PCR amplification. The PCR primers are located in the promoter, leader or framework sequences of the VHH for the 5xe2x80x2 primer and in the hinge, CH2, CH3, 3xe2x80x2 untranslated region or polyA tail for the 3xe2x80x2 primer. A size selection of amplified material allows the construction of a library limited to heavy chain immunoglobulins.
In a particular example, the following 3xe2x80x2 primer in which a KpnI site has been constructed and which corresponds to amino-acids 313 to 319 (CGC CAT CAA GGT AAC AGT TGA) SEQ ID NO: 47 is used in conjunction with mouse VHH primers described by Sestry et al and containing a Xho site
These primers yield a library of camelid heavy chain immunoglobulins comprising the VHH region (related to mouse or human subgroup III), the hinge and a section of CH2.
In another example, the cDNA is polyadenylated at its 5xe2x80x2 end and the mouse specific VHH primers are replaced by a poly T primer with an inbuilt XhoI site, at the level of nucleotide 12.
CTCGAGT12.
The same 3xe2x80x2 primer with a KpnI site is used.
This method generates a library containing all subgroups of immunoglobulins.
Part of the interest in cloning a region encompassing the hinge-CH2 link is that in both xcex32 and xcex33, a Sac site is present immediately after the hinge. This site allows the grafting of the sequence coding for the VHH and the hinge onto the Fc region of other immunoglobulins, in particular the human IgG1 and IgG3 which have the same amino acid sequence at this site (Glu246 Leu247).
As an example, the invention contemplates a cDNA library composed of nucleotide sequences coding for a heavy-chain immunoglobulin, such as obtained by performing the following steps:
a) treating a sample containing lymphoid cells, especially periferal, lymphocytes, spleen cells, lymph nodes or another lyphoid tissue from a healthy animal, especially selected among the Camelids, in order to separate the lymphoid cells,
b) separating polyadenylated RNA from the other nucleic acids and components of the cells,
c) reacting the obtained RNA with a reverse transcriptase in order to obtain the corresponding cDNA,
d) contacting the cDNA of step c) with 5xe2x80x2 primers corresponding to mouse VH domain of four-chain immunoglobulins, which primer contains a determined restriction site, for example an XhoI site and with 3xe2x80x2 primers corresponding to the N-terminal part of a CH2 domain containing a KpnI site,
e) amplifying the DNA,
f) cloning the amplified sequence in a vector, especially in a bluescript vector,
g) recovering the clones hybridizing with a probe corresponding to the sequence coding for a constant domain from an isolated heavy-chain immunoglobulin.
This cloning gives rise to clones containing DNA sequences including the sequence coding for the hinge. It thus permits the characterization of the subclass of the immunoglobulin and the SacI site useful for grafting the FVHHh to the Fc region.
The recovery of the sequences coding for the heavy-chain immunoglobulins can also be achieved by the selection of clones containing DNA sequences having a size compatible with the lack of the CH1 domain.
It is possible according to another embodiment of the invention, to add the following steps between steps c) and d) of the above process:
in the presence of a DNA polymerase and of deoxyribonucleotide triphosphates, contacting said cDNA with oligonucleotide degenerated primers, which sequences are capable of coding for the hinge region and N-terminal VHH domain of an immunoglobulin, the primers being capable of hybridizing with the cDNA and capable of initiating the extension of a DNA sequence complementary to the cDNA used as template,
recovering the amplified DNA.
The clones can be expressed in several types of expression vectors. As an example using a commercially available vector Immuno PBS (Huse et al: Science (1989) 246, 1275), clones produced in Bluescript(copyright) according to the above described procedure, are recovered by PCR using the same XhoI containing 5xe2x80x2 primer and a new 3xe2x80x2 primer, corresponding to residues 113-103 in the framework of the immunoglobulins, in which an Spe site has been constructed: TC TTA ACT AGT GAG GAG ACG GTG ACC TG SEQ ID NO: 51. This procedure allows the cloning of the VHH in the Xho/Spe site of the Immuno PBS vector. However, the 3xe2x80x2 end of the gene is not in phase with the identification xe2x80x9ctagxe2x80x9d and the stop codon of the vector. To achieve this, the construct is cut with Spe and the 4 base overhangs are filled in, using the Klenow fragment after which the vector is religated. A further refinement consists in replacing the marker (xe2x80x9ctagxe2x80x9d) with a poly histidine so that metal purification of the cloned VHH can be performed. To achieve this a Spe/EcoRI double stranded oligo-nucleotide coding for 6 histidines and a termination codon is first constructed by synthesis of both strands followed by heating and annealing:
CTA GTG CAC CAC CAT CAC CAT CAC TAA TAG SEQ ID NO: 52
AC GTG GTG GTA GTG GTA GTG ATT ATC TTA A SEQ ID NO: 53
The vector containing the insert is then digested with SpeI and EcoRI to remove the resident xe2x80x9ctagxe2x80x9d sequence which can be replaced by the poly-His/termination sequence. The produced VHH can equally be detected by using antibodies raised against the dromedary VHH regions. Under laboratory conditions, VHH regions are produced in the Immuno PBS vector in mg amounts per liter.
The invention also relates to a DNA library composed of nucleotide sequences coding for a heavy-chain immunoglobulin, such as obtained from cells with rearranged immunoglobulin genes.
In a preferred embodiment of the invention, the library is prepared from cells from an animal previously immunized against a determined antigen. This allows the selection of antibodies having a preselected specificity for the antigen used for immunization.
In another embodiment of the invention, the amplification of the cDNA is not performed prior to the cloning of the cDNA.
The heavy-chain of the four-chain immunoglobulins remains sequestered in the cell by a chaperon protein (BIP) until it has combined with a light chain. The binding site for the chaperon protein is the CH1 domain. As this domain is absent from the heavy chain immunoglobulins, their secretion is independent of the presence of the BIP protein or of the light chain. Moreover the inventors have shown that the obtained immunoglobulins are not sticky and accordingly will not abnormally aggregate.
The invention also relates to a process for the preparation of a monoclonal antibody directed against a determined antigen, the antigen binding site of the antibody consisting of heavy polypeptide chains and which antibody is further devoid of light polypeptide chains, which process comprises:
immortalizing lymphocytes, obtained for example from the peripheral blood of Camelids previously immunized with a determined antigen, with an immortal cell and preferably with myeloma cells, in order to form a hybridoma,
culturing the immortalized cells (hybridoma) formed and recovering the cells producing the antibodies having the desired specificity.
The preparation of antibodies can also be performed without a previous immunization of Camelids.
According to another process for the preparation of antibodies, the recourse to the technique of the hybridoma cell is not required.
According to such process, antibodies are prepared in vitro and they can be obtained by a process comprising the steps of:
cloning into vectors, especially into phages and more particularly filamentous bacteriophages, DNA or cDNA sequences obtained from lymphocytes especially PBLs of Camelids previously immunized with determined antigens,
transforming prokaryotic cells with the above vectors in conditions allowing the production of the antibodies,
selecting the antibodies for their heavy-chain structure and further by subjecting them to antigen-affinity selection,
recovering the antibodies having the desired specificity,
In another embodiment of the invention the cloning is performed in vectors, especially into plasmids coding for bacterial membrane proteins. Procaryotic cells are then transformed with the above vectors in conditions allowing the expression of antibodies in their membrane.
The positive cells are further selected by antigen affinity selection.
The heavy chain antibodies which do not contain the CH1 domain present a distinct advantage in this respect. Indeed, the CH1 domain binds to BIP type chaperone proteins present within eukaryotic vectors and the heavy chains are not transported out of the endocytoplasmic reticulum unless light chains are present. This means that in eukaryotic cells, efficient cloning of 4-chain immunoglobulins in non mammalian cells such as yeast cells can depend on the properties of the resident BIP type chaperone and can hence be very difficult to achieve. In this respect the heavy chain antibodies of the invention which lack the CH1 domain present a distinctive advantage.
In a preferred embodiment of the invention the cloning can be performed in yeast either for the production of antibodies or for the modification of the metabolism of the yeast. As example, Yep 52 vector can be used. This vector has the origin of replication (ORI) 2xcexc of the yeast together with a selection marker Leu 2.
The cloned gene is under the control of gall promoter and accordingly is inducible by galactose. Moreover, the expression can be repressed by glucose which allows the obtention of very high concentration of cells before the induction.
The cloning between BamHI and SalI sites using the same strategy of production of genes by PCR as the one described above, allows the cloning of camelid immunoglobulin genes in E. coli. As example of metabolic modulation which can be obtained by antibodies and proposed for the yeast, one can site the cloning of antibodies directed against cyclins, that is proteins involved in the regulation of the cellular cycle of the yeast (TIBS 16 430 J. D. Mc Kinney, N. Heintz 1991). Another example is the introduction by genetic engineering of an antibody directed against CD28, which antibody would be inducible (for instance by gall), within the genome of the yeast. The CD28 is involved at the level of the initiation of cell division, and therefore the expression of antibodies against this molecule would allow an efficient control of multiplication of the cells and the optimization of methods for the production in bioreactors or by means of immobilized cells.
In yet another embodiment of the invention, the cloning vector is a plasmid or a eukaryotic virus vector and the cells to be transformed are eukaryotic cells, especially yeast cells, mammalian cells for example CHO cells or simian cells such as Vero cells, insect cells, plant cells, or protozoan cells.
For more details concerning the procedure to be applied in such a case, reference is made to the publication of Marks et al, J. Mol. Biol. 1991, 222:581-597.
Furthermore, starting from the immunoglobulins of the invention, or from fragments thereof, new immunoglobulins or derivatives can be prepared.
Accordingly immunoglobulins replying to the above given definitions can be prepared against determined antigens. Especially the invention provides monoclonal or polyclonal antibodies devoid of light polypeptide chains or antisera containing such antibodies and directed against determined antigens and for example against antigens of pathological agents such as bacteria, viruses or parasites. As example of antigens or antigenic determinants against which antibodies could be prepared, one can cite the envelope glycoproteins of viruses or peptides thereof, such as the external envelope glycoprotein of a HIV virus, the surface antigen of the hepatitis B virus.
Immunoglobulins of the invention can also be directed against a protein, hapten, carbohydrate or nucleic acid.
Particular antibodies according to the invention are directed against the galactosylxcex1-1-3-galactose epitope.
The immunoglobulins of the invention allow further the preparation of combined products such as the combination of the heavy-chain immunoglobulin or a fragment thereof with a toxin, an enzyme, a drug, a hormone.
As example one can prepare the combination of a heavy-chain immunoglobulin bearing an antigen binding site recognizing a myeloma immunoglobulin epitope with the abrin or mistletoe lectin toxin. Such a construct would have its uses in patient specific therapy.
Another advantageous combination is that one can prepare between a heavy-chain immunoglobulins recognizing an insect gut antigen with a toxin specific for insects such as the toxins of the different serotypes of Bacillus thuringiensis or Bacillus sphaericus. Such a construct cloned into plants can be used to increase the specificity or the host range of existing bacterial toxins.
The invention also proposes antibodies having different specificities on each heavy polypeptide chains. These multifunctional, especially bifunctional antibodies could be prepared by combining two heavy chains of immunoglobulins of the invention or one heavy chain of an immunoglobulin of the invention with a fragment of a four-chain model immunoglobulin.
The invention also provides hetero-specific antibodies which can be used for the targetting of drugs or any biological substance like hormones. In particular they can be used to selectively target hormones or cytokines to a limited category of cells. Examples are a combination of a murine or human antibody raised against interleukin 2 (IL2) and a heavy-chain antibody raised against CD4 cells. This could be used to reactivate CD4 cells which have lost their IL2 receptor.
The heavy-chain immunoglobulins of the invention can also be used for the preparation of hetero-specific antibodies. These can be achieved either according to the above described method by reduction of the bridges between the different chains and reoxydation, according to the usual techniques, of two antibodies having different specificities, but it can also be achieved by serial cloning of two antibodies for instance in the Immuno pBS vector.
In such a case, a first gene corresponding to the VHH domain comprised between Xho site and a Spe site is prepared as described above. A second gene is then prepared through an analogous way by using as 5xe2x80x2 extremity a primer containing a Spe site, and as 3xe2x80x2 extremity a primer containing a termination codon and an EcoRI site. The vector is then digested with EcoRI and XhoI and further both VHH genes are digested respectively by Xho/Spe and by Spe/EcoRI.
After ligation, both immunoglobulin genes are serially cloned. The spacing between both genes can be increased by the introduction of addition codons within the 5xe2x80x2 SpeI primer.
In a particular embodiment of the invention, the hinge region of IgG2 immunoglobulins according to the invention is semi-rigid and is thus appropriate for coupling proteins. In such an application proteins or peptides can be linked to various substances, especially to ligands through the hinge region used as spacer. Advantageously the fragment comprises at least 6 amino acids.
According to the invention it is interesting to use a sequence comprising a repeated sequence Pro-X, X being any amino-acid and preferably Gln, Lys or Glu, especially a fragment composed of at least a 3-fold repeat and preferably of a 12-fold repeat, for coupling proteins to ligand, or for assembling different protein domains.
The hinge region or a fragment thereof can also be used for coupling proteins to ligands or for assembling different protein domains.
Usual techniques for the coupling are appropriate and especially reference may be made to the technique of protein engineering by assembling cloned sequences.
The antibodies according to this invention could be used as reagents for the diagnosis in vitro or by imaging techniques. The immunoglobulins of the invention could be labelled with radio-isotopes, chemical or enzymatic markers or chemiluminescent markers.
As example and especially in the case of detection or observation with the immunoglobulins by imaging techniques, a label like technetium, especially technitium 99 is advantageous. This label can be used for direct labelling by a coupling procedure with the immunoglobulins or fragments thereof or for indirect labelling after a step of preparation of a complex with the technitium.
Other interesting radioactive labels are for instance indium and especially indium 111, or iodine, especially I131, I125 and I123.
For the description of these techniques reference is made to the FR patent application published under number 2649488.
In these applications the small size of the VHH fragment is a definitive advantage for penetration into tissue.
The invention also concerns monoclonal antibodies reacting with anti-idiotypes of the above-described antibodies.
The invention also concerns cells or organisms in which heavy-chain immunoglobulins have been cloned. Such cells or organisms can be used for the purpose of producing heavy-chain immunoglobulins having a desired preselected specificity, or corresponding to a particular repertoire. They can also be produced for the purpose of modifying the metabolism of the cell which expresses them. In the case of modification of the metabolism of cells transformed with the sequences coding for heavy-chain immunoglobulins, these produced heavy-chain immunoglobulins are used like antisense DNA. Antisense DNA is usually involved in blocking the expression of certain genes such as for instance the variable surface antigen of trypanosomes or other pathogens. Likewise, the production or the activity of certain proteins or enzymes could be inhibited by expressing antibodies against this protein or enzyme within the same cell.
The invention also relates to a modified 4-chain immunoglobulin or fragments thereof, the VH regions of which has been partialy replaced by specific sequences or amino acids of heavy chain immunoglobulins, especially by sequences of the VHH domain. A particular modified VH domain of a four-chain immunoglobulin, is characterized in that the leucine, proline or glutamine in position 45 of the VH regions has been replaced by other amino acids and preferably by arginine, glutamic acid or cysteine.
A further modified VH or VL domain of a four-chain immunoglobulin, is characterized by linking of CDR loops together or to FW regions by the introduction of paired cysteines, the CDR region being selected between the CDR1 and the CDR3, the FW region being the FW2 region, and especially in which one of the cysteines introduced is in position 31, 33 of the CDR1 or 45 of FW2; and the other in CDR3.
Especially the introduction of paired cysteines is such that the CDR3 loop is linked to the FW2 or CDR1 domain and more especially the cysteine of the CDR3 of the VH is linked to a cysteine in position 31, 33 of the CDR1 or in position 45 of FW2.
In another embodiment of the invention, plant cells can be modified by the heavy-chain immunoglobulins according to the invention, in order that they acquire new properties or increased properties.
The heavy-chain immunoglobulins of the invention can be used for gene therapy of cancer for instance by using antibodies directed against proteins present on the tumor cells.
In such a case, the expression of one or two VHH genes can be obtained by using vectors derived from parvo or adeno viruses. The parvo viruses are characterized by the fact that they are devoid of pathogenicity or almost not pathogenic for normal human cells and by the fact that they are capable of easily multiplying in cancer cells (Russel S. J. 1990, Immunol. Today II. 196-200).
The heavy-chain immunoglobulins are for instance cloned within HindIII/XbaI sites of the infectious plasmid of the murine MVM virus (pMM984). (Merchlinsky et al, 1983, J. Virol. 47, 227-232) and then placed under the control of the MVM38 promoter.
The gene of the VHH domain is amplified by PCR by using a 5xe2x80x2 primer containing an initiation codon and a HindIII site, the 3xe2x80x2 primer containing a termination codon and a XbaI site.
This construct is then inserted between positions 2650 (HindIII) and 4067 (XbaI) within the plasmid.
The efficiency of the cloning can be checked by transfection. The vector containing the antibody is then introduced in permissive cells (NB-E) by transfection.
The cells are recovered after two days and the presence of VHH regions is determined with an ELISA assay by using rabbit antiserum reacting with the VHH part.
The invention further allows the preparation of catalytic antibodies through different ways. The production of antibodies directed against components mimicking activated states of substrates (as example vanadate as component mimicking the activated state of phosphate in order to produce their phosphoesterase activities, phosphonate as compound mimicking the peptidic binding in order to produce proteases) permits to obtain antibodies having a catalytic function. Another way to obtain such antibodies consists in performing a random mutagenesis in clones of antibodies for example by PCR, in introducing abnormal bases during the amplification of clones. These amplified fragments obtained by PCR are then introduced within an appropriate vector for cloning. Their expression at the surface of the bacteria permits the detection by the substrate of clones having the enzymatic activity. These two approaches can of course be combined. Finally, on the basis of the data available on the structure, for example the data obtained by XRay crystallography or NMR, the modifications can be directed. These modifications can be performed by usual techniques of genetic engineering or by complete synthesis. One advantage of the VHH of the heavy chain immunoglobulins of the invention is the fact that they are sufficiently soluble.
The heavy chain immunoglobulins of the invention can further be produced in plant cells, especially in transgenics plants. As example the heavy chain immunoglobulins can be produced in plants using the pMon530 plasmid (Roger et al. Meth Enzym 153 1566 1987) constitutive plant expression vector as has been described for classical four chain antibodies (Hiat et al. Nature 342 76-78, 1989) once again using the appropriate PCR primers as described above, to generate a DNA fragment in the right phase.